Nickel-containing compounds are human carcinogens. The mechanisms of their carcinogenic actions remain to be investigated. Recent studies have indicated that reactive oxygen species (ROS) may play an important role. We hypothesize that nickel induces generation of ROS, which activate nuclear transcription factors, leading to cell transformation and tumorigenesis. Specific Aim 1 will detect and identify ROS generated in human bronchial epithelial cells (BEAS-2B) and mouse embryo fibroblast cells exposed to nickel compounds and investigate the mechanism involved. We hypothesize that nickel (Ni3S2 and NiCl2) can stimulate the cells to activate NADPH oxidase via cdc42 and p47phox to produce superoxide radical, which is then converted to hydrogen peroxide and hydroxyl radical. Specific Aim 2 will test the hypothesis that ROS are required for activation of NFAT and NFkappaB in cells and in vivo in response to nickel compounds. The role of ROS in nickel-induced activation of NFAT and NFkappaB in cells will be evaluated by co-transfection of NFkappaB-luciferase or NFAT-luciferase reporter plasmids and specific ROS scavenger enzymes. For in vivo study, BALB/c transgenic mice with alternation of antioxidant enzymes or NADPH oxidase (ROS generating enzyme) will be employed. Specific Aim 3 will investigate the role of ROS, NFAT and NFkappaB in nickel-induced cell transformation and tumorigenesis. We hypothesize that ROS activate transcription factors and cause cell transformation and tumorigenesis. We will use overexpression of DN-NFAT, DN-IkappaBalpha and DN- IKKbeta to investigate the involvement of NFAT and NFkappaB in nickel-cell transformation and induced tumorigenesis. The role of ROS will be investigated using specific antioxidant enzyme expressions and NADPH oxidase alternation. We anticipate that nickel causes activation of NFAT and NFkappaB through ROS reactions, leading to cell transformation and tumorigenesis. We attempt to link the cell transformation and tumorigenesis with specific transcription factors and specific reactive oxygen species. The results obtained from this proposal will elucidate the role of ROS and NFAT/NFkappaB signaling in Ni compounds-induced carcinogenesis. The long term goals are to provide a fundamental understanding concerning the mechanism of carcinogenic actions of Ni;to fill a need for the mechanistic information of cancer risk assessment for exposure;to propose methods for early detection;and to develop intervention and prevention strategies. Nickel-containing compounds are human carcinogens. This project will investigate the mechanism of Ni-induced carcinogenesis by testing the hypothesis that nickel induces generation of ROS, which activate nuclear transcription factors, leading to cell transformation and tumorigenesis. The long term goals are to understand the mechanism of Ni-carcinogenesis;to propose methods for early detection;and to develop intervention and prevention strategies.